Insulin is a peptide synthesized, stored and secreted by the .beta. cells present in the pancreatic islets of Langerhans [Orci, L. The insulin cell: its cellular environment and how it processes (pro) insulin. Diabetes Metab. Rev. 2:71-106, 1986]. Insulin is a hormone with a wide array of biological activities primarily targeted at the liver, muscle and fat tissues. Through its interaction with cellular receptors, insulin activates systems involved in the intracellular utilization and storage of glucose, amino acids and fatty acids, as well as inhibiting catabolic processes that result in the breakdown of glycogen, protein and fat (Kahn, C. R. and White, M. F. The insulin receptor and the molecular mechanism of insulin action. J. Clin. Invest. 82:1151-1156, 1988). Under normal physiological conditions the presence of elevated levels of glucose in the blood leads to insulin secretion in a highly regulated process. Although glucose is the principal stimulus of insulin secretion in humans, its ingestion as part of the food components induces the release of other gastrointestinal hormones that also participate in the promotion of insulin secretion (Meglasson, M. D. and Matschinsky, F. M. Pancreatic islet glucose metabolism and regulation of insulin secretion. Diabetes Metab. Rev. 2:163-214, 1986). Blood glucose, however, is sufficient for the induction of insulin secretion in two phases--a short-lived, rapid 1 to 2 minutes first phase, and a longer phase of a delayed onset. The mechanism of insulin release by glucose is not fully understood, but is known to require the entry of glucose into pancreatic .beta. cells and a metabolism within these cells (see Meglasson and Matschinsky, 1986, supra).
Diabetes mellitus is a complex set of diseases that are characterized by high blood glucose levels (hyperglycemia) and altered carbohydrate, lipid and protein metabolism leading to clinical complications from vascular disorders, eye disorders such as retinopathy, glaucoma and cataracts, nephropathy, diabetic neuropathy and a variety of infections. In addition, several serious acute diabetic complications such as diabetic ketoacidosis and lactic acidosis can occur, and often with lethal consequences--especially in the elderly (Pirart, J. Diabetes mellitus and its degeneration complications: prospective study of 4,400 patients observed between 1947 and 1973. Diabetes Care 1:168-188, 1978). Most patients can be clinically classified as having either insulin-dependent diabetes mellitus (IDDM or Type-I diabetes) or non-insulin-dependent diabetes mellitus (NIDDM or Type-II diabetes). Diabetes mellitus, or glucose intolerance (GIT), is one of the major diseases in the United States, afflicting mostly the elderly population (it is estimated that 7% of men and 9% of women over the age of 65 years have diabetes mellitus). In the United States about 90% of diabetic patients have Type-II diabetes and most of the remainder suffer from Type-I diabetes. There is evidence that Type-I diabetes is an autoimmune type disease of the pancreatic .beta. cells with a continuous destruction of pancreatic .beta. cells and increased inability to synthesize and secrete insulin (Srikanta, S. et al. Type-I diabetes mellitus in monozygotic twins: chronic progressive .beta. cell dysfunction. Ann. Intern. Med. 99:320-326, 1983). In Type-II diabetes there is no significant loss of .beta. cells from pancreatic islets, and although the .beta. cells retain their ability to synthesize and secrete insulin their ability to respond to a glucose challenge is diminished, especially through the first phase of insulin secretion (Genuth, S. Plasma insulin and glucose profiles in normal, obese, and diabetic persons. Ann. Intern. Med. 79:812-822, 1973; Leahy, J. L. et al. Chronic hyperglycemia is associated with impaired glucose influence on insulin secretion: a study in normal rats using chronic in vivo glucose infusions. J. Clin. Invest. 77:908-915, 1987).
Insulin is the mainstay treatment for essentially all Type-I and many Type-II diabetic patients. Elevated blood levels of insulin produce immediate inhibition of liver glucose production (by glycogenolysis and gluconeogenesis) and a marked stimulation of uptake and metabolism of glucose by muscle and adipose tissue, leading to the overall reduction in blood glucose levels. Insulin treatment is classified into short, intermediate or long acting, depending on the preparation of insulin and its mode of delivery (intravenous vs. intramuscular vs. subcutaneous injections). Many Type-II diabetic patients can be adequately treated by diet or hypoglycemic agents other than insulin. The overwhelming adverse reaction to insulin is hypoglycemia. Hypoglycemia (or low blood glucose) is a major risk and it should be weighed against the benefits of insulin treatment, especially in Type-II diabetes patients where increased plasma insulin can be achieved by proper diet or milder hypoglycemic drugs, since the pancreatic .beta. cells are still capable of secreting insulin in this disease. The oral hypoglycemic agents that are suitable for Type-II diabetic patients are divided into the sulfonylureas and biguanides as well as other oral hypoglycemic agents that are not structurally related. For recent reviews of current antidiabetic drugs, see: Krall, L. P. Oral hypoglycemic agents. In Joslin's Diabetes Mellitus, 12th Ed. (Marble, A., et al., Editors) Lea & Febiger, Philadelphia, 1985, pp 412-452; and Kahn, C. R. and Shechter, Y. Insulin, oral hypoglycemic agents, and the pharmacology of the endocrine pancreas. In Goodman & Gilman's The Pharmacological Basis of Therapeutics, 8th Ed. (Gilman, A. G. et al., Editors), Pergamon Press, Inc., 1990, pp 1463-1495. Since many diabetic patients, in particular the elderly, suffer from impaired hepatic and renal functions, their ability to metabolize and excrete the synthetic hypoglycemic agents is severely limited, leading to serious adverse reactions.
More than 10 years ago, two studies clearly demonstrated that extracellular ATP and certain analogues of ATP can induce insulin secretion from isolated perfused animal pancreas (Loubati eres-Mariani, M. M, Chapal, J., Lignon, F. and Valett, G. Structural specificity of nucleotides for insulin secretory action from the isolated perfused rat pancreas. Eur. J. Pharmacol. 59:277-286, 1979; and Chapal, J. and Loubati eres-Mariani, M. M. Effects of phosphate-modified adenine nucleotide analogues on insulin secretion from perfused rat pancreas. Br. J. Pharmacol. 73:105-110, 1981). More recently it has been shown that insulin secretion can be stimulated by the interaction of P.sub.2y -purine receptor agonists with the P.sub.2y -purine receptors present on the surface of pancreatic cells and that synthetic agonists such as adenosine-5'-0-(2-thiodiphosphate) are one hundred times more active than ATP in stimulating insulin secretion in vitro in perfused rat pancreas (Bertrand, G. et al. Adenosine-5'-0-(2-thiodiphosphate) is a potent agonist of P.sub.2 -purinoceptors mediating insulin secretion from perfused rat pancreas. Br. J. Pharmacol. 102:627-630, 1991). The same P.sub.2y -purine receptor agonist was very recently shown to be effective in stimulating insulin secretion and improving glucose tolerance in rats and dogs in vivo (Hilliare-Buys, D. et al. Stimulation of insulin secretion and improvement of glucose tolerance in rat and dog by the P.sub.2y -purinoceptor agonist, adenosine 5'-0-(2-thiodiphosphate). Br. J. Pharmacol. 109:183-187, 1993). Thus, the purine receptor agonists that were selected for the studies of stimulation of insulin secretion by pancreatic cells in vitro or in vivo are analogues of ATP such as 2-methylthio ATP, which is a much more powerful agonist for the P.sub.2y receptor than is ATP (Ribes, G. et al. Effects of 2-methylthio ATP on insulin secretion in the dog in vivo. Eur. J. Pharmacol. 155:171-174, 1988) or analogues of ATP that are both stronger agonists with increased chemical and biological stability, such as adenosine 5'-0-(2-thiodiphosphate) (Bertrand, G. et al., 1991, supra; Hilliare-Buys, D. et al., 1993 supra). These recent reports rule out ATP itself as a potential agent for the stimulation of insulin secretion for the stated reasons: in vivo instability due to rapid metabolism and poor agonist properties for the P.sub.2y -purine receptor.
U. S. Pat. No. 4,880,918 entitled "Arrest and Killing of Tumor Cells by Adenosine 5'-Diphosphate and Adenosine 5'-Triphosphate" to Rapaport, U.S. Pat. No. 5,049,372 entitled "Anticancer Activities in a Host by Increasing Blood and Plasma Adenosine 5'-Triphosphate (ATP) Levels" to Rapaport, and U.S. Pat. No. 5,227,371 entitled "Utilization of Adenine Nucleotides and/or Adenosine and Inorganic Phosphate for Elevation of Liver, Blood and Blood Plasma Adenosine 5'-Triphosphate Concentrations" to Rapaport, disclose the treatment of cancer by administration of adenine nucleotides to a human host and/or disclose a method to expand organ, blood and blood plasma ATP pools by the administration of adenine nucleotides and/or adenosine and inorganic phosphate to a human host.
The role of intracellular ATP as a cellular energy source, a phosphate group donor for phosphorylation reactions and an allosteric regulator of the activities of a variety of cellular proteins has been well-established. Only in the past 10 years have the roles of adenosine and ATP began to emerge as powerful physiological extracellular modulators of intravascular, extravascular and CNS functions, a role which is attracting significant attention within the field of drug development (Williams, M. Purinergic drugs: opportunities in the 1990's. Drug Devel. Res. 28:438-444, 1993). Adenosine is the endogenous ligand for the A (or P.sub.1) type purine receptors affecting mostly cardiovascular and CNS functions, whereas ATP is the ligand for P.sub.2 type purine receptors and is now an accepted neurotransmitter (Benham, C. D. ATP joins the fast lane. Nature 359:103-104, 1992; Edwards, F. A., Gibb, A. J. and Colquhoun, D. ATP receptor-mediated synaptic currents in the central nervous system. Nature 359:144-147, 1992).
The administration of adenine nucleotides (e.g., ATP, AMP or other adenine nucleotides) into the systemic circulation results in the immediate degradation of the nucleotide to adenosine and inorganic phosphate. This degradation in the vascular bed is followed by incorporation of the adenosine and inorganic phosphate into liver ATP pools (steady state levels) yielding significant expansion of the liver ATP pools, which is followed by an expansion of red blood cell ATP pools. The red blood cells with expanded ATP pools which are produced by this mechanism slowly release micromolar levels of ATP into the blood plasma without undergoing hemolysis, thus achieving elevated steady state extracellular ATP levels, in spite of the catabolic enzymatic activities present intravascularly (Rapaport, E. and Fontaine, J. Anticancer activities of adenine nucleotides in mice are mediated through the expansion of erythrocyte ATP pools. Proc. Natl. Acad. Sci. USA 86:1662-1666, 1989). These elevated levels of ATP inhibit both tumor growth and host weight loss in tumor-bearing murine models. The inhibition of tumor growth proceeds by the receptor-mediated and non-receptor-mediated effects of extracellular ATP on the tumor cell membrane, whereas the inhibition of host weight loss in tumor-bearing hosts is the result of ATP-mediated marked slowdown of hepatic gluconeogenesis and reversal of the depletion of visceral energy stores (Rapaport, E. Mechanisms of anticancer activities of adenine nucleotides in tumor-bearing hosts. Ann. NY Acad. Sci. 603:142-150, 1990).
Administration of ATP by intravenous infusions at a dose of 50 .mu.g/kg min for at least 48 hours yielded a doubling of blood (red blood cell) ATP levels after 24 hours in advanced cancer patients (most of whom were at stage IIIB or IV non-small cell lung cancer). Hyperuricemia developed only after at least 48 hours of continuous infusions (Haskell, C. M. and Sanchez-Anaya, D. Hyperuricemia as a complication of ATP: preliminary observation of a phase I clinical trial. ASCO Proc. 12:435A, 1993)and could be easily dealt with by administering allopurinol. The elevated blood ATP levels declined within several days after termination of the ATP infusions with a return of total blood ATP levels to their basal levels. In advanced cancer patients with cachexia and malnutrition, the basal blood ATP levels were lower than normal but could be elevated to well above a normal level after ATP infusions.
The mechanisms of expansion of organ ATP levels after administration of ATP proceed by both the increased supply of the major purine precursor for salvage ATP synthesis in cells (adenosine), and by the interaction of extracellular ATP with membrane P.sub.2 -purine receptors which signals an enhanced intracellular ATP synthesis. Most of the expansions of total blood (red blood cell) ATP pools occur due to increased supply of purines to the mature erythrocyte in the hepatic sinusoids, where these purine precursors (mostly adenosine) arise from the increases in turnover of hepatic ATP pools (Rapaport, E. and Fontaine, J. Generation of extracellular ATP in blood and its mediated inhibition of host weight loss in tumor-bearing mice. Biochem. Pharmacol. 38:4261-4266, 1989). A significant increase in red blood cell ATP pools of the magnitude observed in vivo after ATP administration cannot be obtained in vitro (Rapaport, E. and Fontaine, J. Anticancer activities of adenine nucleotides in mice are mediated through expansion of erythrocyte ATP pools. Proc. Natl. Acad. Sci. USA 86:1662-1666, 1989).
Adenosine 5'-triphosphate (ATP) infusions useful against metastatic refractory cancers are in phase I of human clinical trials. The two questions which are being answered by these trials are: 1) is it possible to achieve the degree of elevation of red blood cells and blood plasma compartment pools of ATP after the administration of ATP to patients as was shown extensively in preclinical murine models, and 2) can the elevated ATP levels in the human host produce the spectrum of anticancer activities demonstrated in experimental animals (Rapaport, E. Mechanisms of anticancer activities of adenine nucleotides in tumor-bearing hosts. Ann. NY Acad. Sci. 603:142-150, 1990).
A variety of in vitro and in vivo studies have demonstrated several anticancer activities of extracellular (blood plasma compartment) pools of ATP as well as elevated hepatic and red blood cell pools of ATP. These activities are a) cytostatic and cytotoxic effects on the tumor; b) anticachexia effects and improvement in hepatic and renal functions; c) modulation of tumoral blood flow; d) antianemia effects; e) antipain activities; f) improvements in motor functions, performance status; g) improvements in oxygen delivery to peripheral sites; h) enhancement of superoxide anion (O.sub.2) production by phagocytic cells, and i) significant antithrombotic effects in vivo. All of these anticancer activities observed either in experimental animals or in humans after the administration of ATP have been reviewed recently (Rapaport, E. Anticancer activities of adenine nucleotides in tumor-bearing hosts. Drug Devel. Res. 28:428-431, 1993).
The administration of ATP to tumor-bearing murine hosts was also shown to markedly inhibit host weight loss in a cachectic tumor model and, as importantly, the administration of ATP or other adenine nucleotides was shown to elevate extracellular, blood plasma compartment steady state levels (pools) of ATP. The inhibition of tumor growth and host weight loss were shown not to exhibit a cause and effect relationship in murine models.
Whereas ATP itself has been shown a long time ago to stimulate insulin secretion from the pancreatic .beta. cells of the isolated perfused rat pancreas (Loubati eres-Mariani, et al., 1979, supra; Chapal and Loubati eres-Mariani, 1981, Supra), extensive studies that followed these initial reports demonstrated that only synthetic analogues of ATP can be considered for stimulating insulin secretion in vivo. These analogues include 2-methylthio ATP, a powerful agonist of the P.sub.2y -purine receptor which is 45 times more potent than ATP in increasing insulin secretion in in vitro systems (Bertrand, G. et al. Evidence for two different P.sub.2 -purinoceptors on .beta. cell and pancreatic vascular bed. Br. J. Pharmacol. 91:783-787, 1987), and which was demonstrated to be effective in the dog by its direct infusion into the pancreaticoduodenal artery (Ribes, G., 1988, supra). The other (and even more powerful) analogue of ATP is the chemically and biologically stable adenosine-5'-0-(2-thiodiphosphate) which is also a strong P.sub.2y -purine receptor agonist and was shown to be 100 times more potent than ATP in the in vitro insulin secretion system of the isolated perfused pancreas of the rat (Bertrand, G. et al., 1991, supra). Adenosine-5'-0-(2-thiodiphosphate) was then used successfully in the rat and dog in vivo in an injectable or oral delivery system for stimulation of insulin secretion (Hilliare-Buys, D. et al., 1993, supra). Thus, all the previous data teach away from ATP itself being utilized as an insulin secretagogue in vivo in a human suffering from glucose intolerance. The widely accepted notion is that ATP is a weak P.sub.2y -purine receptor (the extracellular receptor involved in the regulation of insulin secretion) agonist as compared to other available synthetic ATP analogues. More importantly, the state of the art teaches that because of the rapid degradation of ATP in vivo, only a chemically and biologically stable analogue of ATP would provide a sufficient agonist concentration at the P.sub.2y -purine receptors of the pancreatic .beta. cells to affect insulin secretion in a therapeutically meaningful way.